Method for determining decrease in functions of hippocampus by using correlation between micro rna and nmda receptor, method for inhibiting decrease in functions, and method for screening for inhibitors of decrease in functions

ABSTRACT

Provided are a method for determining a decrease in the functions of the hippocampus by using the correlation between a micro RNA (miRNA) and an N-methyl-D-aspartate receptor (NMDAR), a method for inhibiting the decrease in the functions, and a method for screening for inhibitors of the decrease in the functions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation in part application of U.S. patent application Ser. No. 16/889,372 filed on Jun. 1, 2020, which is a Divisional Application of U.S. patent application Ser. No. 15/736,949 filed on Dec. 15, 2017, which is a National Stage application of PCT/KR2016/006465 filed on Jun. 17, 2016, which claims priority to Korean Patent Application No. 10-2015-0086823 filed on Jun. 18, 2015, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND 1. Field of the Invention

The present disclosure relates to a method for determining decrease in functions of hippocampus using correlation between micro RNA (mRNA) and N-methyl-D-aspartate receptor (NMDA receptor; NMDAR), a method for inhibiting decrease in functions, and a method for screening for inhibitors of decrease in functions, more particular, since as the expression of miRNA-204 increases in the aged hippocampus, the Eph/ephrin mediated axon guidance signaling pathway is down-regulated, which causes the expression of the hippocampal NMDA receptor is decreased, the present disclosure relates to determine that whether hippocampal function is decreased by using the correlation thereof, furthermore, to provide a method for inhibiting decrease in functions of the hippocampus and a method for inhibitors of decrease in functions.

2. Discussion of Related Art

The hippocampus is a key component of the brain system and is part of the cerebral cortex that protrudes from the floor of lateral ventricle inferior horn in the cerebrum. The hippocampus regulates learning, memory and awareness of new things, emotions, actions and some movements, and regulates the functions of the hypothalamus. The hippocampus above is undergoing biological and structural changes with age and affect age-related cognitive decline (C. A. Erickson and C. A. Barnes, Experimental Gerontology, 38:61, 2003). Neurocontrol defects caused by aging can affect the memory and learning abilities of the aged hippocampus, which can lead to Alzheimer's dementia, epilepsy, and Clover-Verse syndrome (Ryann M. Fame et al., Trends Neurosci. 34(1):41, 2011; Federico W. Grillo et al., PNAS, 110(16):E1514, 2013).

In particular, the brain is formed by a network of neuron (nerve cell) connected by synapses, and our memory information is generated in such a way that the synapses, the connection structure between neurons, are changed. When a gene related to memory is expressed and a protein is made, memory contents are preserved and maintained for a long time, and synaptic plasticity changes in aging process. The synaptic plasticity implies a structural or biochemical change in synapses that changes the effect of post-synaptic neurons, and electrical stimulation of circuits in the hippocampus can induce long-term synaptic changes that appear to be associated with learning. N-methyl-D-aspartate receptor (NMDA receptor), a neuro-receptor known to regulate signaling of between cell death and normal cell, are found in the hippocampus, especially in the CA1 region, the NMDA receptor is known to regulate calcium throughout the body and to participate in long-term enhancement.

In addition, since the NMDA receptors interact directly with the dopamine D1 receptor and regulate cell death according to the result of the interaction or induce normal cell-to-cell communication, various studies have been conducted on NMDA receptors to treat various diseases such as stroke, schizophrenia, osteoporosis, epilepsy, and dementia. For example, It is a well-established academic theory that the blackout phenomenon, a phenomenon in which alcohol cannot be remembered after drinking alcohol, also occurs because alcohol acts on the brain to interrupt the activity of NMDA receptors. In other words, the above is because when NMDA activity is interrupted, the glutamate salt activity, which acts as a mediator between neurons in the brain, is stopped.

On the other hand, microRNA (miRNA) is a new material that binds to 3′-UTR of mRNA as a single-stranded RNA molecule of 21-25 nucleotides (nt) and regulates expression in eukaryotes. The production of the miRNA is made into a pre-miRNA of stemloop structure by Drosha (RNase III type enzyme), the pre-miRNA is made into miRNA by being transported to the cytoplasm and cleaved by Dicer. The miRNA is involved in development, cell proliferation and death, fat metabolism, tumor formation, and the like by regulating the expression of target proteins. The miRNAs having the function above have recently attracted attention as other epigenetic regulators other than histone deacetylases (HDACs) or DNA methyl transferases (DNMTs) (Miranda K C et al., Cell, 126(6):1203, 2006; Nicole Noren Hooten et al., PLoS ONE, 5(5):e10724, 2010).

It is well-known that about 50% of all mammalian miRNAs are abundant in the brain and many of the miRNAs have an important role in nerve and neural development (Sempere, L. F. et al., Genome Biol., 5:R13, 2004; Giraldez, A. J. et al., Science, 308:833, 2005).

In the prior art, although it is reported as miRNAs in the entire brain of rat are potentially involved in the regulation of the aging process through the insulin signaling pathway (Sachi Inukai et al., PLoS ONE, 7(7):e40028, 2012), and it is known as regulation of neural activity is performed by interaction of downstream targets in the hippocampal miRNAs and the particular biological pathway (Stephen M. Eacker et al., PLoS ONE, 6(10):e25068, 2011), there is still a lack of comprehensive research on the effect of miRNA dynamic expression on normal hippocampal function in an aging-dependent manner.

Accordingly, the present inventors have made extensive efforts to study the role of miRNA in the normal aging process of the hippocampus, it was confirmed that of the miRNAs up-regulated in the aging hippocampus, miR-204 accurately targets EphB2 an important regulatory receptor and reduces EphB2 expression, and which causes neuronal surface expression of the NR1 subunit of the NMDA receptors in hippocampal nerves was decreased and dendritic density decreased was resulted in.

In addition, the present inventors determined that whether hippocampal function was decreased by using the correlation between microRNA and NMDA receptor, furthermore, confirmed that a method for inhibiting decrease in functions of the hippocampus and a method for screening inhibitors of decrease in functions can be provided, thus completed the present disclosure.

SUMMARY OF THE INVENTION

An object of the present disclosure is to provide a method for determining decrease in functions of hippocampus using correlation between micro RNA (mRNA) and N-methyl-D-aspartate receptor (NMDA receptor; NMDAR).

Another object of the present disclosure is to provide a method for inhibiting decrease in functions of hippocampus using correlation between miRNA and NMDA receptor.

Another object of the present disclosure is to provide a use for using of an agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p for preventing or treating decrease in functions of hippocampus.

Another object of the present disclosure is to provide a method for preventing decrease in functions of hippocampus, comprising: administering an agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p to an individual in need thereof in a pharmaceutically effective amount.

Another object of the present disclosure is to provide a method for treating decrease in functions of hippocampus, comprising: administering an agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p to an individual in need thereof in a pharmaceutically effective amount.

Another object of the present disclosure is to provide a method for screening for inhibitors of decrease in functions of hippocampus using correlation between miRNA and NMDA receptor.

The present disclosure, to achieve the first object, provides a method for determining decrease in functions of hippocampus, which includes: (a) analyzing an expression of a miR-204-5p represented by a base sequence of SEQ ID NO: 1 in a gene sample collected from a patient; and (b) determining whether the expression of the miR-204-5p from the patient is increased by using a gene sample collected from a normal person as a control group; according to increasing the expression of the miR-204-5p, further determining whether an expression of N-methyl-D-aspartate receptor (NMDA receptor; NMDAR) on a surface of a hippocampal nerves is decreased.

According to a preferred embodiment of the present disclosure, as the expression of the miR-204-5p is increased, an expression of an EphB2 protein may be decreased, and resulting in a decrease in an expression of a NMDA receptor in the hippocampal nerves.

According to a preferred embodiment of the present disclosure, as the expression of miR-204-5p is increased, an expression of a NR1 subunit may be decreased.

According to a preferred embodiment of the present disclosure, as the expression of the miR-204-5p gene is increased, a dendritic spine density of the hippocampal nerve may be decreased.

The present disclosure, to achieve the second object, provides a method for inhibiting decrease in functions of hippocampus, which includes: increasing an expression of a NMDA receptor by treating an agent that inhibits an expression of miR-204-5p or inhibits an activity of miR-204-5p.

According to a preferred embodiment of the present disclosure, the agent which inhibits the expression of the miR-204-5p or inhibits the activity of the miR-204-5p may be an antisense oligonucleotide or a siRNA.

According to a preferred embodiment of the present disclosure, the agent may be at least one selected from the group consisting of an antisense RNA 5′AGGCAUAGGAUGACAAAGGGAA 3′.

According to a preferred embodiment of the present disclosure, as an expression of an EphB2 protein may be increased by the agent which inhibits the expression of the miR-204-5p or inhibits the activity of the miR-204-5p and resulting in a decrease in an expression of a NMDA receptor in a hippocampal nerves.

According to a preferred embodiment of the present disclosure, of the NMDA receptors, NR1 subunit expression may be increased.

According to a preferred embodiment of the present disclosure, a dendritic spine density of the hippocampal nerves may be increased by the agent that inhibits the expression of miR-204-5p or inhibits an activity of a miR-204-5p.

The present disclosure, to achieve the third object, provides a method for screening an inhibitor of decrease in functions of hippocampus, which include: (a) treating a candidate which inhibits a miR-204-5p expression or inhibits an activity of a miR-204-5p in a human-derived hippocampal nerve; and (b) determining whether an expression of a NMDA receptor is increased on a surface of the nerve.

According to a preferred embodiment of the present disclosure, in the step (b), the candidate which increases the expression of the NMDA receptor may be selected.

According to a preferred embodiment of the present disclosure, in the step (b), further determining whether an expression of EphB2 protein increases, and the candidate which increases the expression of the EphB2 protein and the expression of the NMDA receptor may be selected.

According to a preferred embodiment of the present disclosure, in the step (b), whether the expression increased of the NR1 subunit in the NMDA receptor may be determined.

Further, the present disclosure may provide to a pharmaceutical composition for preventing or treating decrease in functions of hippocampus, comprising: an agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p, wherein miR-204-5p is represented by the base sequence of SEQ ID NO: 1.

In addition, the present disclosure may provide to a use of an agent which inhibits an expression of miR-204-5p or inhibits an activity of miR-204-5p for preventing or treating decrease in functions of hippocampus, wherein the miR-204-5p is represented by a base sequence of SEQ ID NO: 1.

In addition, the present disclosure may provide to a method for preventing decrease in functions of hippocampus, which includes: administering an agent which inhibits an expression of miR-204-5p or inhibits an activity of miR-204-5p to an individual in need thereof in a pharmaceutically effective amount, wherein the miR-204-5p is represented by a base sequence of SEQ ID NO: 1.

Further, the present disclosure may provide to a method for treating decrease in functions of hippocampus, which includes: administering an agent which inhibits an expression of miR-204-5p or inhibits an activity of miR-204-5p to an individual in need thereof in a pharmaceutically effective amount, wherein the miR-204-5p is represented by a base sequence of SEQ ID NO: 1.

One aspect is to provide a method of treating cognitive impairment or Alzheimer's disease, the method including administering an effective amount of an agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p to an individual in need thereof.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may be a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p.

In an embodiment, the nucleic acid molecule may be RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2′-0 modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate-backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may include the oligonucleotide sequence of SEQ ID NO: 1.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may include a vector containing a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p, or a cell transformed thereby.

In an embodiment, the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p may be included in a viral vector.

In an embodiment, the viral vector may be an AAV vector.

Another aspect is to provide a method of inhibiting amyloid beta, the method including administering an effective amount of an agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p to an individual in need thereof.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may be a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p.

In an embodiment, the nucleic acid molecule may be RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2′-0 modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate-backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may include the oligonucleotide sequence of SEQ ID NO: 1.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may include a vector containing a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p, or a cell transformed thereby.

In an embodiment, the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p may be included in a viral vector.

In an embodiment, the viral vector may be an AAV vector.

In the present disclosure, since it was confirmed that of the miRNAs up-regulated in the aging hippocampus, miR-204 accurately targets EphB2 an important regulatory receptor and reduces EphB2 expression, and which causes neuronal surface expression of the NR1 subunit of the NMDA receptors in hippocampal nerves was decreased and dendritic density decreased was resulted in, there are effects of determining whether hippocampal function was decreased by using the correlation between microRNA and NMDA receptor, furthermore, providing a method for inhibiting decrease in functions of the hippocampus and a method for screening inhibitors of decrease in functions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an aging-dependent expression pattern of hippocampal mRNA, and illustrates a heat map of expression change of miRNA in hippocampus of 2 months, 6 months, and 18 months rats, miRNA with a fold change value of greater than 2.0 is about 80.

FIG. 1B illustrates a scattered volume plot of miRNA having a fold change value of greater than 2.0 between 2 months and 18 months rats, Representative miRNAs selected through qPCR assays are represented in red. The X axis represents a volume change and the Y axis represents an miRNA expression as the log the fold change value.

FIG. 1C illustrates a graph of data which represents the types of miRNAs having a fold change value of greater than 2.0 between 2 months and 18 months rats when analysis is performed using Real-Time Quantitative Polymerase Chain Reaction (qPCR).

FIG. 2A illustrates modeled data a predicted pathway of Eph/ephrin mediated axon induction in hippocampal aging, and qPCR-verified targets are represented with #.

FIG. 2B illustrates data of a fold change value of a representative target mRNA included in the axon induction path using qPCR.

FIG. 2C illustrates data of a fold change value of target mRNA involved in Eph/ephrin signal in hippocampal tissue using qPCR.

FIG. 2D illustrates data of protein expression of target molecules involved in ephrin signaling in young hippocampal tissue and old hippocampal tissue determined by Western blot analysis.

FIG. 2E shows a graph which represents the protein expression of target molecules involved in ephrin signaling in young hippocampal tissue and old hippocampal tissue.

FIG. 3 illustrates data on the selection of miRNAs involved in EphB2.

FIG. 4 illustrates data of relative RNA expression of miRNA204-5p and EphB2.

FIG. 5A illustrates EphB2 which is a target of miR-204-5p in neuron, and illustrates relative luciferase activity when miR-204-5p was treated with wild-type and mutated EphB2-3′UTR.

FIG. 5B illustrates data of the luciferase activity of miR-204-5p concentration-dependent EphB2-3′UTR in cultured hippocampal nerves.

FIG. 5C illustrates data determined by western blotting of expression change of EphB2 when neurons were treated with miR-204 mimic and miR-204 inhibitor.

FIG. 5D shows a graph which represents the expression change of EphB2 when neurons were treated with miR-204 mimic and miR-204 inhibitor.

FIG. 6A illustrates data determined of whether a dendritic density is regulated by miR-204.

FIG. 6B shows a graph which represents the dendritic density regulated by miR-204.

FIG. 7A illustrates data of surface expression degree of NR1 subunit of the NMDA receptor by miR-204. The biotinylated (surface) NR1 and total NR1 proteins in hippocampal nerves transformed with scramble (scr) on day 7 of culture, miR-204 mimic and miR-204 inhibitor were visualized by immunostaining using specific antibodies. Tubulin was used as a loading control group of the total protein, and the absence of tubulin indicates the effectiveness of the surface label.

FIG. 7B shows a graph which represents the expression ratios of surface NR1 and total NR1 on day 7 of culture (*P<0.05, error bars: standard mean error).

FIG. 7C illustrates data quantifying protein expression degree of the NMDA receptor NR1 subunit using western blot analysis from young and old hippocampal tissue. Protein band quantification and relative density were analyzed using image J software.

FIG. 7D shows a graph which represents the expression ratio of NR1 from old hippocampal tissue and NR1 from young hippocampal tissue.

FIG. 8A shows an image of a mir-204 inhibitor injection group, from which the decrease in amyloid plaques has been confirmed.

FIG. 8B shows a graph of a mir-204 inhibitor injection group, from which the decrease in amyloid plaques has been confirmed. FIG. 9 shows a graph of a mir-204 inhibitor injection group, from which the decrease in human amyloid plaques has been confirmed.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the present invention is described in more detail.

As described above, although micro RNA (miRNA, hereinafter referred to as miRNA) in brain are known to play important roles in nerve and neural development, there is a lack of research on how the miRNA affects normal hippocampal function in an aging-dependent manner, depending on a regulation of miRNA expression.

The present disclosure, sought to solve the above-mentioned problems by providing a method for determining decrease in functions of hippocampus using correlation between micro RNA and NMDA receptor, a method for inhibiting decrease in functions, and a method for screening inhibitors of decrease in functions. Through the above, it was confirmed that of the miRNAs up-regulated in aged hippocampus, miR-204 reduces EphB2 expression by correctly targeting EphB2 which is an important regulatory receptor, which causes the expression of NR1 subunit of the NMDA receptor was decreased on the surface of the hippocampal nerve cell and dendritic density decrease was resulted in.

Accordingly, the present disclosure relates to a method for determining decrease in functions of hippocampus, comprises,

(a) analyzing an expression of a miR-204-5p represented by a base sequence of SEQ ID NO: 1 in a gene sample collected from the patient;

(b) determining whether the expression of the miR-204-5p from the patient is increased by using a gene sample collected from a normal person as a control group;

according to increasing the expression of the miR-204-5p, further determining whether an expression of N-methyl-D-aspartate receptor (NMDA receptor; NMDAR) on a surface of a hippocampal nerves is decreased.

In addition, the present disclosure can provide a method for diagnosing memory impairment and a method for cognitive function inhibition using the method for determining decrease in functions of the hippocampus, further can provide a method for diagnosing dementia.

In the present disclosure, it is characterized that when the expression of EphB2 protein is decreased as the expression of the miR-204-5p is increased, the Eph/ephrin mediated axon guidance signaling pathway is down-regulated, which causes the expression of NMDA receptor in the hippocampal nerves is decreased, and preferably, it is characterized that of NMDA receptor on the cell surface of neuron (nerve cell), the expression of the NR1 subunit decreases.

In addition, it is characterized that as the expression of miR-204-5p gene is increased, the dendritic spine density in the hippocampal nerves is decreased.

The miR-204-5p expressed by the base sequence of SEQ ID NO: 1 (5′-uucccuuugucauccuaugccu-3′) was known to inhibit the growth of cancer cells mainly (Binbin Zhang et al., Medical Oncology, 32(1):1, 2014), however, it was first confirmed by the present inventors that miR-204 reduces EphB2 expression by correctly targeting EphB2, which causes expression of the NR1 subunit of the NMDA receptor expressed on the surface in the hippocampal nerves is decreased cell and dendritic density decrease was resulted in.

The miR-204 or miR-204-5p nucleic acid molecule of the present disclosure can be originated from an animal including a human, for example, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, and the like, but it is not limited thereto. In addition, the miR-204 or the miR-204-5p nucleic acid molecule of the present disclosure can exist in a single-stranded or double-stranded form. Although mature miRNA molecule exists predominantly single-stranded, precursor miRNA (pre-miRNA) molecule exists predominantly self-complementary (for example, stem- and loop-structure) structure capable of forming double-stranded portion.

The nucleic acid molecule of the present disclosure can obtain information through a known sequence information database, for example, GenBank of NCBI, through above, may be separated or prepared by using standard molecular biology techniques, for example, chemical synthesis methods or recombinant methods, or commercially available ones can be used.

In one embodiment of the present disclosure, to understand the miRNA mediating a regulation of a hippocampal aging process, small RNA (small RNA; smRNA or sRNA, hereinafter referred to as sRNA) profiling was performed in male rats at 2 months, 6 months and 18 months. The sRNA is an RNA consisting of approximately 200 bases or less, which corresponds to eukaryotic cells and is known to exclude tRNA and ribosomal RNA.

As shown in FIG. 1A, a heat map analysis was performed to confirm miRNAs that were differentially expressed as the aging progressed, and of the total 269 miRNAs in the hippocampus, 80 miRNAs were confirmed to have fold change values greater than 2.0 for any two of the three different time periods. miRNAs showed significant increase in expression or decrease in expression, especially, The presence of miRNAs with significantly increased expression in a mouse of 18 months was confirmed.

As shown in FIG. 1B, in the sporadic volume configuration of 269 miRNAs expressed in the hippocampus during normal aging (between 2 months and 18 months), it was confirmed that 19 miRNAs were decreased more than 2-fold in expression, on the other hand, 36 miRNAs were increased more than 2-fold in expression. Also, to confirm the significance of sRNA data, twelve miRNAs having fold change values of 2.0 or greater in rats of 2 and 18 months were randomly selected (increase in expression: 5, decrease in expression: 7) and analyzed by real-time quantitative polymerase chain reaction (real-time quantitative PCR; qPCR). As shown in FIG. 1C, it was confirmed that the result were consistent with sRNA profiling data and sRNA profiling can reflect the entire miRNA expression pattern in hippocampal tissue.

In the present disclosure, the expected target gene pathway of miRNAs was derived to determine the biological effects caused by miRNA expression regulation in hippocampal aging. As shown in Table 2, various biological effects were derived, although the cancer-related pathway was expected to be significantly significant, the follow-up study was carried out with a focus on the axon guidance pathway (P=4.5E-18), which has relatively little research in the aspect of aging. As the schematic diagram of the axon guidance pathway illustrated in FIG. 2A, in the present disclosure, the relationship between the activity of Eph/ephrin and miRNA was examined.

As shown in FIG. 2B, it was confirmed in the present disclosure that of the target molecules in the Eph/ephrin subfamily, the expression of 9 molecules were decreased in the aging process, no significant change was observed in 13 molecules, the expression of was homolog gene family (RhoA, member A) was only increased, using qPCR. This above shows down-regulation of Eph/ephrin mediated axon induction pathway by aging, on the whole. In particular, EphA1 and EphB2, two ephrin receptors known to regulate synaptic plasticity, were found to be approximately 30% and 25% decreased in mRNA expression, respectively.

To confirm down-regulation of ephrin signal pathway by aging, as shown in FIG. 2C, the fold change values of the target mRNAs involved in the Eph/ephrin signal in hippocampal tissues of 2 and 18 months rats were analyzed using qPCR. The results of the analysis generally corresponded to the sRNA profile analysis results of the present disclosure, it was confirmed that EphrinB3, which was a single ephrin subunit, and EphA1, EphA2, EphA4 and EphB2, which were four ephrin receptor subunits, were decreased mRNA expression by 25 to 60%, on the other hand, RhoA mRNA was increased expression by 50%.

In addition, to confirm whether the protein expression of the above molecules also changed with aging, the degree of protein expression for RhoA, EphA4 and EphB2 was confirmed, as a result, as shown in FIGS. 2D and 2E, it was confirmed that EphA4 and EphB2 was decreased protein expression by 60% and 30%, respectively, RhoA was increased protein expression up to 150%.

In the present disclosure, EphB2 which is one of the positive regulators that regulate synaptic plasticity in the ephrin signal pathway, is important for neuroplasticity and is known to cause cognitive dysfunction in Alzheimer's disease, was selected as a target of miRNA which expression is significantly regulated in the aging period.

As shown in FIG. 3, in the present disclosure, 8 candidate miRNAs, which are miR-24-3p, miR-34C-5p, miR-204-5p, miR-346-5p, miR-495-3p, miR149-5p, miR-485-5p, miR-383-5p, having a binding site for 3′UTR (SEQ ID NO: 2) of EphB2 and having a decrease in expression more than 2-fold in 2 and 18 months rats were confirmed (MIMAT0000219, MIMAT0000381, MIMAT0000237, MIMAT0000597, MIMAT0003456, MIMAT0000159, MIMAT0003128, MIMAT0000748), and it was confirmed that of them, only miR-204-5p represented by the base sequence of SEQ ID NO: 1 inhibits EphB2 activity. In addition, as shown in FIG. 4, it was confirmed that mRNA expression of miR-204-5p and EphB2 are inversely correlated as aging progresses.

In one embodiment of the present disclosure, to confirm whether miR-204-5p was specific to EphB2, the activities of wild-type EphB2-3′UTR-WT (AGGATTCTCATAAGGGAA) and miR-204 binding site point mutated EphB2-3′UTR-MT (AGGATTCTCATGGAGAGA) were compared. As shown in FIG. 5A, EphB2-3′UTR-WT and miR-204-5p were reacted, as a result, it was confirmed that the activity of EphB2 was abruptly decreased, on the other hand, that of EphB2-3′UTR-MT was not changed. In addition, as shown in FIG. 5B, it was confirmed that the activity of EphB2 was decreased depending as a miR-204-5p concentration-dependent manner.

FIGS. 5C and 5D illustrate, data confirmed changes in EphB2 protein expression by western blot when neurons were treated with miR-204 mimic and miR-204 inhibitor (5′AGGCAUAGGAUGACAAAGGGAA 3′), it was confirmed that the expression amount of EphB2 was significantly decreased to 50% by miR-204, and when the miR-204 inhibitor was treated, it was confirmed that the expression amount of EphB2 was recovered to 130%. In other words, it was confirmed that EphB2 was a direct target of miR-204-5p in the present disclosure.

In one embodiment of the present disclosure based on the above results, the relationship between miR-204-5p and EphB2 in synaptic plasticity was examined. As shown in FIGS. 6A and 6B, the regulation degree of dendritic spine density by miR-204-5p was confirmed, as a result, it was confirmed that the over-expression of miR-204-5p decreased the spine density by 40% when compared with the control group scramble mimic (3.4 spine/10 um length, including 5.2 spine protrusion, p<0.001). When EphB2-3′UTR-WT and miR-204 mimic were simultaneously transfected (3.9 spine/10 um length, p>0.5), it was confirmed that there was no significant increase in spine density compared to miR-204 mimic (3.6 spine/10 um length, p<0.001) only infected neurons, on the other hand, when EphB2-3′UTR-MT and miR-204 mimic were simultaneously transfected (5.4 spine/10 nm length, p<0.001), it was confirmed that there was spine density of normal levels. In other words, the above result means that miR-204-5p regulates the dendritic spine density through the inhibition of EphB2.

In addition, since EphB2 is known to mediate NMDA receptors expressed on the cell surface of neurons, whether miR-204-5p affects the transport and location change of NMDA receptor subunits in the hippocampal nerves were examined.

FIGS. 7A and 7B illustrate data showing the surface expression regulation degree of NR1 subunit of NMDA receptor by miR-204, as a result of confirming the ratio of the cell surface NR1 protein/total NR1 protein, it was confirmed that the expression ratio of NR1 protein was significantly decreased on neuronal cell surface by miR-204, on the other hand, it was confirmed that the expression ratio of NR1 protein was significantly increased by the miR-204 inhibitor.

In addition, as shown in FIGS. 7C and 7D, as results of confirming the expression of the NMDA receptor NR1 subunit from the young (of 2 months mouse) and the old (of months mouse) hippocampal tissues, It was confirmed that the expression of total NR1 protein was decreased by about 50% in the aging process. The above means that the expression of neurons' cell surface and total NR1 protein was decreased as the expression of EphB2 in neurons is decreased by miR-204.

In other words, since it was confirmed in the present disclosure that of the miRNAs up-regulated in the aging hippocampus, miR-204 accurately targets EphB2 an important regulatory receptor and educes EphB2 expression, and which causes neuronal surface expression of the NR1 subunit of the NMDA receptors in the hippocampal nerves to decrease and dendritic density resulting in a decrease, it was confirmed that the correlation between miR-204-5p and EphB2 or the correlation between miR-204-5p and NMDA receptor can be used to determine whether hippocampal function is reduced.

Further, the correlation between the miR-204-5p and EphB2 or the correlation between the miR-204-5p and the NMDA receptor can be utilized in a method for inhibiting decrease in functions of the hippocampus and a method for screening inhibitors of decrease in functions.

In other words, the present disclosure also relates to a method for inhibiting decrease in functions of hippocampus comprising: treating an agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p to increase an expression of a NMDA receptor.

The method can be applied to a decrease in hippocampal function caused by a decrease in an expression of a NMDA receptor or a general aging process.

In addition, the present disclosure can be applied to cognitive function improvement, memory damage inhibition/improvement, the treatment of various diseases resulting from decrease in hippocampal function.

The agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p may be an antisense oligonucleotide or an siRNA. In the present disclosure, preferably miR-204-5p represented by the base sequence of SEQ ID NO: 4 was used as an inhibitor, but oligonucleotides having a complementary sequence to miR-204-5p represented by the base sequence of SEQ ID NO: 1 can be used without limitation, and oligonucleotides capable of selectively binding only specific regions of miR-204-5p that specifically recognize 3′UTR of EphB2 can also be used without limitation.

It is characterized that the expression of EphB2 protein is increased by an agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p, which causes the expression of NMDA receptor in the hippocampal nerves increases. Preferably, it is characterized by an increase in the expression of the NR1 subunit among the NMDA receptor on the cell surface of neuron (nerve cell). In addition, it is characterized by the dendritic spine density in the hippocampal nerves is increased by the agent which inhibits the expression of miR-204-5p or inhibits an activity of miR-204-5p.

As described above, it was confirmed that since the expression of EphB2 protein was increased by miR-204-5p inhibitor and the expression of NR1 subunit was increased in FIG. 5C and FIG. 7A, it was confirmed that the agent inhibiting an expression of miR-204-5p or inhibiting an activity of a miR-204-5p was treated to inhibit decrease in functions of the hippocampus.

The present disclosure also relates to a method for screening a decrease in functions of hippocampus inhibitor comprising: (a) treating a candidate substance which inhibits miR-204-5p expression or inhibits activity of miR-204-5p in human-derived hippocampal nerves; and (b) determining whether expression of the NMDA receptor is increased on the surface of the neuron.

It is characterized that of the candidate substances, a substance that increases the expression of the NMDA receptor is selected. In particular, it can be confirmed whether the expression of the NR1 subunit is increased in the NMDA receptor.

In addition, in step (b), by further confirming the increased expression of EphB2 protein, a candidate substance which increases the expression of EphB2 protein can be selected.

Further, the present disclosure relates to a pharmaceutical composition for preventing or treating decrease in functions of hippocampus, comprising: an agent which inhibits the expression of miR-204-5p represented by the base sequence of SEQ ID NO: 1 or inhibits an activity of miR-204-5p.

In addition, the present disclosure provides to a use for using of an agent which inhibits the expression of miR-204-5p represented by the base sequence of SEQ ID NO: 1 or inhibits an activity of miR-204-5p for preventing or treating decrease in functions of hippocampus.

In addition, the present disclosure provides to a method for preventing decrease in functions of hippocampus, comprising: administering an agent which inhibits the expression of miR-204-5p represented by the base sequence of SEQ ID NO: 1 or inhibits an activity of miR-204-5p to an individual in need thereof in a pharmaceutically effective amount.

Further, the present disclosure provides to a method for treating decrease in functions of hippocampus, comprising: administering an agent which inhibits the expression of miR-204-5p represented by the base sequence of SEQ ID NO: 1 or inhibits an activity of miR-204-5p to an individual in need thereof in a pharmaceutically effective amount.

The composition of the present disclosure can Improve cognitive impairment, and prevent and treat various diseases related to abnormality of hippocampal function due to aging. In addition, since the composition of the present disclosure inhibits an activity of miR-204-5p and increases the expression of NMDA receptor which is a surface protein of hippocampal nerves, it can be applied to the prevention or treatment of various diseases such as stroke, schizophrenia and the like, which are caused by decrease in an expression of a NMDA receptor or abnormal function.

The pharmaceutical composition comprising an agent which inhibits the expression of miR-204-5p represented by the base sequence of SEQ ID NO: 1 or inhibits an activity of miR-204-5p for preventing and treating decrease in functions of hippocampus according to the present disclosure may further include other agents capable of improving hippocampal function.

Another aspect is to provide a method of treating cognitive impairment or Alzheimer's disease, the method including administering, to an individual in need thereof, an effective amount of an agent which inhibits expression of miR-204-5p or an activity of miR-204-5p.

Another aspect is to provide a method of inhibiting amyloid beta, the method including administering an effective amount of an agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p to an individual in need thereof.

The term “agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p” used herein includes any substance capable of inhibiting the expression and/or activity of miR-204-5p. In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may be a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p. In an embodiment, the nucleic acid molecule may be RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2′-0 modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate-backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide.

The term “nucleic acid” used herein includes polynucleotides, oligonucleotides, DNA, RNA, and analogs and derivatives thereof, and includes, for example, peptide nucleic acids (PNA) or mixtures thereof. In addition, the nucleic acid may be single-stranded or double-stranded, and may encode a molecule including a polypeptide, mRNA, microRNA, or siRNA.

In an embodiment, the nucleic acid molecule used herein includes a sequence complementary to all or part of the seed sequence of miR-204-5p. The seed sequence is a sequence which is very important for the recognition of the target molecule of miRNA and is conserved in various species. Since miRNA binds to the target through the seed sequence, when the interaction between the seed sequence and the target is inhibited, the translation of the target mRNA, for example, may be effectively inhibited.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may include the oligonucleotide sequence of SEQ ID NO: 1.

The term “cognitive impairment” used herein refers to disorders in which cognitive ability is impaired or behavioral changes occur compared to those of animals with normal functions, and may include mild cognitive impairment.

In an embodiment, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p may include a vector containing the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p, or a cell transformed thereby.

The term “vector” refers to a means for expressing a target nucleic acid molecule in a host cell. Examples thereof include viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenoviral vectors, retroviral vectors, RNA expression vectors, and adeno-associated viral vectors. Vectors that can be used as the recombinant vector may be prepared by manipulating plasmids that are often used in the art (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), phage or virus (for example, SV40, etc.). In addition, for example, the adeno-associated viral vector may be any one selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

The AAV vector may include an AAV genome or a fragment or derivative thereof. The AAV genome is a polynucleotide sequence, which encodes the functions required for the production of AAV particles. These functions include capsidization of AAV genome into AAV particles, and include actions in the cycle of replication and packaging of AAV in host cells. Naturally occurring AAVs are replication-deficient and rely on the provision of in trans-helper functions to complete the replication and packaging cycle. The AAV genome may be a positive or negative sense single-stranded form. In an embodiment, the AAV genome may be a positive or negative sense double-stranded form. The use of the double-stranded form allows the DNA replication in the target cell to be bypassed, thereby accelerating transgene expression.

The AAV genome may be obtained from any naturally derived serotype, isolate or Glade of AAV. Thus, the AAV genome may be a complete genome of a naturally occurring AAV. As known in the art, naturally occurring AAVs may be classified according to a variety of biological systems. In general, AAV is referred to in terms of their serotype. Serotypes correspond to variant subspecies of AAV, which, due to the expression profile of the capsid surface antigen, has a unique reactivity that can be used to differentiate one variant subspecies from other variant subspecies. Typically, viruses with a specific AAV serotype do not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include recombinant serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV11, and Rec2 and Rec3 which are recently identified in primate brains. Any of these AAV serotypes may be used in the present disclosure.

AAV may also be referred to in terms of clads or clones. This refers to the phylogenetic relationship of naturally-derived AAV, and typically refers to the phylogenetic group of AAVs that may be traced back to a common ancestor, and includes all progeny thereof. Additionally, AAV may be referred to in terms of a particular isolate, that is, a genetic isolate of a particular AAV found in the nature. The term ‘genetic isolate’ defines a separate population that is cognitive at a genetic level by describing the population of AAV that undergoes limited genetic blending with other naturally occurring AAV.

Those skilled in the art may select an appropriate serotype, clad, clone or isolate of AAV for use in the present disclosure based on their common general knowledge.

The adeno-associated virus (AAV) vector is produced by introducing materials capable of making a virus into a specific cell, and An expression vector containing a nucleic acid according to the present disclosure may be introduced into cells by, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells. For example, an expression vector is produced by inserting a gene that inhibits the expression or activity of miR-204-5p into AAV, and then, the expression vector is transduced into a packaging cell, and the transduced packaging cell is cultured and then filtered to obtain an AAV solution. The AAV solution is used to infect cells such as brain cells, nerve cells and/or neural progenitor cells. In this way, the gene that inhibits the expression or activity of miR-204-5p is introduced into cells.

The vector including AAV may refer to a means for expressing a target gene in a host cell. The vector includes elements for expressing the target gene, and may include a replication origin, a promoter, an operator, a terminator, and the like. the vector, and The vector may further include an appropriate enzyme site (for example, a restriction enzyme site) for the introduction into the genome of a host cell, and/or a selectable marker to confirm successful introduction into a host cell and/or a ribosome binding site (RBS) for translation into a protein, internal ribosome entry site (IRES), and the like. The vector may further contain transcriptional control sequences (for example, enhancers, etc.) other than the promoter.

A polynucleotide sequence encoding the protein in the recombinant vector may be operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleotide expression control sequence, such as a promoter sequence, and another nucleotide sequence. Due to the functional linkage, the nucleotide expression control sequence is used for transcription and/or translation of the other nucleotide sequence.

The recombinant vector may be an expression vector capable of stably expressing, in a host cell, the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector may be constructed through various methods known in the art.

In addition, the vector may refer to an expression vector for gene therapy. Accordingly, the viral vector may refer to a viral vector capable of delivering a therapeutic gene or genetic material to a target cell, tissue, and/or organ. The term ‘gene therapy’ refers to a method of normalizing the function of a cell by inserting a normal gene into a cell having a gene abnormality or providing a new function thereto in order to treat various genetic diseases caused by a genetic abnormality.

In an embodiment, the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p may be included in a viral vector. In an embodiment, the viral vector may be AAV vector.

The sequence of miR-204-5p used herein may be derived from mammals, for example, human, mice, or rats.

The inhibition of the expression or activity of miR-204-5p, used herein, refers to inhibition or interference of the intracellular action or function of miR-204-5p, and may include, typically, the direct inhibition of the binding of miR-204-5p to a target molecule thereof, or direct inhibition of the function of miR-204-5p by using a small molecule inhibitor, or an antibody or a fragment thereof, or indirect regulation by using inhibitors or small interfering RNA molecules. Furthermore, the interfering or inhibiting of the activity of miR-204-5p includes directly or indirectly inhibiting the activity of SEQ ID NO: 1. In addition, the inhibiting of the activity of miR-204-5p includes inhibiting the transcription of miR-204-5p to lower the intracellular concentration thereof.

Hereinafter, the present disclosure will be described in detail, which can be readily carried out by a person having ordinary skills in the art to which the present disclosure pertains, with reference to the preferred embodiments. However, the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[Example 1] Analysis of Small RNA (Small RNA) Profiling Related to Hippocampal Aging Process

In the present disclosure, small RNA (smRNA or sRNA, hereinafter referred to as sRNA) profiling was performed in male rats at 2 months, 6 months and 18 months to understand the miRNA mediating the regulation of the hippocampal aging process.

1-1: Animal Preparation and Tissue Collection

General wild-type male C57BL/6J rats of three different ages, 2 months, 6 months and 18 months, were obtained from Korea Research Institute of Bioscience & Biotechnology-Institutional animal care and use committee (KRIBB-IACUC)

Normal rats without tumor or disease signs sacrifices, and hippocampal tissues were immediately dissected from therefrom and stored frozen in liquid nitrogen. Health status of the normal rats was confirmed through a series of serological tests and eight hippocampal tissues were obtained from four rats at each stage. All animals were maintained under “specific pathogens free (SPF)” conditions and all animal studies were performed according to the approval guidelines provided by KRIBB-IACUC.

1-2: Preparation of RNA

RNA extraction and a quality control method were performed using standard protocols.

First, the frozen tissues were homogenized and then extracted with phenol. The obtained RNA pellet was dissolved in nuclease free water and stored at −80° C. until use.

RNA purity and concentration were analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). All samples showed a A260/A280 ratio of 2 or higher and an A260/A230 ratio of 1.9 or higher in nuclease free water.

RNA integrity was analyzed using a Bio-analyzer Nano 6000 chip (Agilent Technologies, USA) to confirm the quality of the RNA. RNA samples with correct 18S/28S and RNA Integrity Number (RIN) values higher than 8 were used for further experiments.

1-3: Extraction of Small RNA and Construction of cDNA Library

Small RNA (hereinafter referred to as small RNA) was extracted using the Pure Link Micro-to-Midi Total RNA Purification System kit (Invitrogen, USA) and miRNeasy mini kit (Qiagen, USA). The sRNA was purified at all stages of rat hippocampal tissue and prepared for Illumina sequencing according to manufacturer's recommendations.

At each stage, 10 μg of sRNA isolated from rat hippocampal tissues of 4 rats was subjected to electrophoresis on a 15% TBE-urea PAGE gel. Then, the fraction of the line containing the RNA molecule which length of the sRNA is between 18 and 30 nucleotides was cleaved from the gel, and the RNA was separated from the gel fragment and ethanol precipitated. The separated sRNA was synthesized as cDNA by attaching 5′ and 3′ adapters (illumina truseq kit, cat No: FC-121-3001) and reverse transcription according to manufacturer's protocol. The obtained cDNA was subjected to PCR amplification using an illumina primer (illumina truseq kit, cat No: FC-121-3001), and the PCR amplification product was electrophoresed and cleaved from the polyacrylamide gel, precipitated with ethanol and separated. Each cDNA library was sequenced using an illuminance sequence analysis technique.

1-4: Deep Sequencing—Next-Generation Sequencing

In the present disclosure, miRNA profiles for three stages of rat hippocampal tissues were characterized using Illumina deep sequencing Hi-seq2000.

In all age groups, the sRNA sample library performed single end sequencing with a read length (length that can be read at one time) of 50 nt (nucleotide). The index base sequence and the 3′ adapter base sequence were removed from the reading, and the sequence containing the adapter dimers, mitochondria or ribosome sequence was discarded. In addition, the sequence those containing homopolymers or shorter than 14 nt were removed from reading.

The results of the readings were subjected to align raw sequences using a software program of cufflink (Johns Hopkins University) and Top-Hat (Johns Hopkins University).

After mapping with reference to the murine genome (Mus_musculus.NCBIM37.55), the sequence read number corresponding to known miRNA to confirm the type of mature miRNA was determined by the sequence that perfectly matched the database of known miRNAs (miR Base release version 16, http://www.mirbase.org/).

1-5: Analysis of miRNA Expression Profile

Raw data (read for each miRNA) was normalized to reads per million (RPM, miRNA counts/total counts of each sample×1 million) and less than 10 readings of miRNA for each sample. In addition, 1 is added to the RPM value to facilitate Log 2 conversion.

The filtered data were transformed by logarithm, differentially expressed miRNA was regulated the difference between 2 months group at the time of regulate and 6 months and 18 months groups to Fold change >|2.0|.

As a result, as shown in FIG. 1A, it was confirmed that of the total 269 miRNAs in the hippocampus, 80 miRNAs exhibit fold change values of 2.0 or higher for any two of the three different time periods.

As shown in FIG. 1B, when sporadic volume configurations of 269 miRNAs in hippocampus expressed during normal aging progression (between 2 months and 18 months), 19 miRNAs were decreased more than 2-fold in expression, on the other hand, 36 miRNAs were increased more than 2 fold in expression.

1-6: Real-Time Quantitative PCR Assay

To confirm the significance of sRNA data, twelve miRNAs having fold change values of 2.0 or greater in rats of 2 and 18 months were randomly selected (increase in expression: 5, decrease in expression: 7) and analyzed by real-time quantitative polymerase chain reaction (real-time quantitative PCR; qPCR).

Selected miRNAs are mmu-miR-191-5p, mmu-miR-342-3p, mmu-miR-99b-5p, mmu-miR-139-5p, mmu-miR-433-3p, mmu-miR-1839-5p, mmu-miR-144-5p, mmu-miR-125b-1-3p, mmu-miR-125b-2-3p, mmu-miR-298-5p and mmu-miR-411-5p. (MIMAT0000221, MIMAT0000590, MIMAT0000132, MIMAT0000656, MIMAT0001420, MIMAT0009456, MIMAT0016988, MIMAT0004669, MIMAT0004529, MIMAT0000376, MIMAT0004747)

First, Invitrogen NCode™ miRNA First strand cDNA module was used to synthesize cDNA by reverse transcription mature miRNA in total RNA isolated from the above.

Total RNA and miRNA (1 μg each) were used to generate cDNA templates according to the manufacturer's instructions. The reaction solution was reacted in a Bio-Rad Thermal Cycler (Bio-Rad, USA) at a total reaction volume of 20 μl. PCR was performed using Bio-Rad real-time PCR system (CFX-96), Each primer design for miRNA was performed using Primer Blast interface (http://www.ncbi.nlm nih gov/tools/primer-blast/) and PrimerBank (http://pga.mgh.harvard.edu/primerbank/). All reactions were performed by dispensing into two sets (using universal reverse primers for miRNAs) of 96-well plates with the SYBR Green Mix and each primer set.

TABLE 1 Sequence Target (5′-3′) SEQ ID NO. mmu-miR- forward CAACGGAAUCC SEQ ID NO: 5 191-5p CAAAAGCAGCUG mmu-miR- forward UCUCACACAGA SEQ ID NO: 6 342-3p AAUCGCACCCGU mmu-miR- forward CACCCGUAGAA SEQ ID NO: 7 99b-5p CCGACCUUGCG mmu-miR- forward UCUACAGUGCA SEQ ID NO: 8 139-5p CGUGUCUCCAG mmu-miR- forward AUCAUGAUGGG SEQ ID NO: 9 433-3p CUCCUCGGUGU mmu-miR- forward AAGGUAGAUAG SEQ ID NO: 10 1839-5p AACAGGUCUUG mmu-miR- forward GGAUAUCAUCA SEQ ID NO: 11 144-5p UAUACUGUAAGU mmu-miR- forward ACGGGUUAGGC SEQ ID NO: 12 125b-1-3p UCUUGGGAGCU mmu-miR- forward ACAAGUCAGGU SEQ ID NO: 13 125b-2-3p UCUUGGGACCU mmu-miR- forward GGCAGAGGAGG SEQ ID NO: 14 298-5p GCUGUUCUUCCC mmu-miR- forward UAGUAGACCGU SEQ ID NO: 15 411-5p AUAGCGUACG universal reverse GTGCAGGGTCC SEQ ID NO: 16 miRNA GAGGT

As a result, as shown in FIG. 1C, it was confirmed that it coincided with the sRNA profiling data, it was confirmed that sRNA profiling can reflect the entire miRNA expression pattern in hippocampal tissue.

[Example 2] Pathway Analysis of Target Gene

All lists of expected target genes for differentially expressed miRNAs according to the aging process were entered into gene functional analysis (http://david.abcc.ncifcrf.gov/) of DAVID, Kyoto Encyclopedia of a Genes and Genomes (KEGG) and Ingenuity Pathway Analysis (Ingenuity® Systems, http://www.ingenuity.com) with limited basic parameters for the results of rat species.

TABLE 2 Gene P- Benjamin Tern Count Value Score Pathway in 136 2.7E−23 4.9E−21 cancer Axon 69 4.5E−18 4.1E−06 quidance Insulin 67 3.7E−15 2.2E−13 signaling pathway Regulation of 89 2.3E−14 1.0E−12 actin cytoskeleton Focal 83 4.6E−14 1.7E−12 adhesion Colorectal 49 3.6E−13 1.1E−11 cancer Adherens 43 9.0E−13 2.3E−11 junction MAPK 98 1.8E−12 4.1E−11 signaling pathway Wntsignaling 63 5.1E−11 1.0E−9  pathway Endocytosis 75 8.7E−10 1.6E−8 

As a result, various biological effects were derived as shown in Table 2, cancer-related pathways were expected to be significantly present, however the follow-up study was carried out with a focus on the axon guidance pathway (P=4.5E-18), which has relatively little research in the aspect of aging. As the schematic diagram of the axon guidance pathway illustrated in FIG. 2A, in the present disclosure, the relationship between the activity of Eph/ephrin and miRNA was examined.

[Example 3] Determination of Eph/Ephrin Pathway Regulation by Aging

3-1: Determination of mRNA Expression

In the present disclosure, qPCR analysis was performed to examine how the expression of Eph/ephrin subfamily changes by aging.

First, PrimeScript™ RT reagent Kit (Promega ImPromm-II Reverse Transcription System) was used to synthesize cDNA by reverse transcription of RNA of the rat hippocampal tissues of each stage separated in Example 1. Total RNA (1 μg each) was used to generate cDNA templates according to the manufacturer's instructions. The reaction solution was reacted in a Bio-Rad Thermal Cycler (Bio-Rad, USA) at a total reaction volume of 20 μl.

PCR was performed using Bio-Rad real-time PCR system (CFX-96), Each primer design for miRNA was performed using Primer Blast interface (http://www.ncbi nlm nih.gov/tools/primer-blast/) and PrimerBank (http://pga.mgh.harvard.edu/primerbank/). All reactions were performed by dispensing into two sets of 96-well plates with the SYBR Green Mix and each primer set.

TABLE 3 Target Sequence (5′-3′) SEQ ID NO. EphA1 forward AGGAAGTCACTCTAATGGACACA SEQ ID NO: 17 reverse CCTCACTCCACCCAGTCTCT SEQ ID NO: 18 EphA2 forward GCACAGGGAAAGGAAGTTGTT SEQ ID NO: 19 reverse CATGTAGATAGGCATGTCGTCC SEQ ID NO: 20 EphA3 forward CATGGGTGGGAAGAGATCAGT SEQ ID NO: 21 reverse GATGCTCTCGGAATTTGACGC SEQ ID NO: 22 EphA4 forward TGGAATTTGCGACGCTGTCA SEQ ID NO: 23 reverse CACTTCCTCCCACCCTCCTT SEQ ID NO: 24 EphB1 forward CCTCCTCCTATGGACTGCCC SEQ ID NO: 25 reverse AAGGCCGTGAAGTCTGGGATA SEQ ID NO: 26 EphB2 forward GCGGCTACGACGAGAACAT SEQ ID NO: 27 reverse GGCTAAGTCAAAATCAGCCTCA SEQ ID NO: 28 EphB3 forward CATGGACACGAAATGGGTGAC SEQ ID NO: 29 reverse GCGGATAGGATTCATGGCTTCA SEQ ID NO: 30 EphrinA1 forward CTTCACGCCTTTTATCTTGGGC SEQ ID NO: 31 reverse TGGGGATTATGAGTGATTTTGCC SEQ ID NO: 32 EphrinA2 forward CGATACGCAGTCTACTGGAAC SEQ ID NO: 33 reverse GGTAGTCGTTGATGCTCACCT SEQ ID NO: 34 EphrinA3 forward CGTGCAGGTGAACGTGAAC SEQ ID NO: 35 reverse GTAACGCTGGAACTTCTCGGA SEQ ID NO: 36 EphrinB1 forward TGTGGCTATGGTCGTGCTG SEQ ID NO: 37 reverse CCAAGCCCTTCCCACTTAGG SEQ ID NO: 38 EphrinB2 forward ATTATTTGCCCCAAAGTGGACTC SEQ ID NO: 39 reverse GCAGCGGGGTATTCTCCTTC SEQ ID NO: 40 EphrinB3 forward TGCTGCTGTTAGGTTTTGCG SEQ ID NO: 41 reverse CTGAGGATAAAGCACGTAACCG SEQ ID NO: 42 Rgs forward AGTGCAGGATGCAGGTCAAC SEQ ID NO: 43 reverse GAAAGAAGAAGTGCTCGTGGAA SEQ ID NO: 44 RhoA forward AGCTTGTGGTAAGACATGCTTG SEQ ID NO: 45 reverse GTGTCCCATAAAGCCAACTCTAC SEQ ID NO: 46 Rock2 forward TTGGTTCGTCATAAGGCATCAC SEQ ID NO: 47 reverse TGTTGGCAAAGGCCATAATATCT SEQ ID NO: 48 cdk5 forward CCCTGAGATTGTGAAGTCATTCC SEQ ID NO: 49 reverse CCAATTTCAACTCCCCATTCCT SEQ ID NO: 50 Mapk1 forward GGTTGTTCCCAAATGCTGACT SEQ ID NO: 51 reverse CAACTTCAATCCTCTTGTGAGGG SEQ ID NO: 52 Rasa1 forward TGTGGTGATTACTACATTGGTGG SEQ ID NO: 53 reverse CGCCTTCTATCTTCTACTGGCTC SEQ ID NO: 54 Pak2 forward AACGGAGAGCTAGAAGACAAGC SEQ ID NO: 55 reverse TGGAACAGAAGGCAAAGGTTT SEQ ID NO: 56 Fyn forward ACCTCCATCCCGAACTACAAC SEQ ID NO: 57 reverse CGCCACAAACAGTGTCACTC SEQ ID NO: 58 Met forward GTGAACATGAAGTATCAGCTCCC SEQ ID NO: 59 reverse TGTAGTTTGTGGCTCCGAGAT SEQ ID NO: 60 Actin forward GGCTGTATTCCCCTCCATCG SEQ ID NO: 61 reverse CCAGTTGGTAACAATGCCATGT SEQ ID NO: 62

As a result, as shown in FIG. 2B, it was confirmed in the aging process that 9 molecules of the target molecules in the Eph/ephrin subfamily were reduced in expression, 13 molecules of the target molecules were not observed any significant change, the expression of was homolog gene family (RhoA, member A) was only increased. In particular, EphA1 and EphB2, two ephrin receptors known to regulate synaptic plasticity, were found to be approximately 30% and 25% decreased in mRNA expression, respectively.

In addition, as shown in FIG. 2C, the fold change values of the target mRNAs involved in the Eph/ephrin signal in hippocampal tissues of 2 and 18 months rats were analyzed using qPCR. As a result, it was confirmed that EphrinB3, which was a single ephrin subunit, and EphA1, EphA2, EphA4 and EphB2, which were four ephrin receptor subunits, were decreased mRNA expression by 25 to 60% (p<0.05), on the other hand, RhoA mRNA was increased expression by 50% (p<0.05).

3-2: Determination of Protein Expression

To confirm whether the protein expression of the above molecules also changed with aging, the degree of protein expression for RhoA, EphA4 and EphB2 was confirmed.

Western blots were performed on each hippocampal tissue sample according to the Abcam® Western blotting protocol. First, 20 to 30 μg of the total lysate was taken, electrophoresed on a 5% sodium dodecyl sulfate/5-20% gradient polyacrylamide gel (SDS-PAGE), and electro-transferred to a nitrocellulose membrane.

The blots were hybridized with first antibody and then treated with second antibody, and each antibody was diluted in a Tris-buffered saline solution containing 5% dry milk. Hybridized bands were confirmed using an enhanced chemiluminescence (ECL) detection system (Describe the used ECL solution and measuring instrument information).

α-Tubulin (AbClon; AbC-2001), RhoA (abcam; ab68826), EphB2 (R&D systems; H00002048-M03) and EphA4 (Abnova; H00002043-M02) were used as the first antibodies and a peroxidase-conjugated second antibody (Jackson ImmunoResearch Laboratories) was used.

Protein band expression of western blot was quantified using image-J software (ImageJ 1.46).

As a result, as shown in FIGS. 2D and 2E, EphA4 (p<0.005, n=4) and EphB2 (p<0.005, n=4) decreased protein expression by 60% and 30%, respectively, RhoA(P=0.005, n=4) increased protein expression up to 150%.

[Example 4] Selection of miRNA Targeting EphB2

The present disclosure seeks to select miRNA targeting EphB2, of which expression is markedly regulated in the aging period

First, 8 candidate miRNAs, which are miR-24-3p, miR-34C-5p, miR-204-5p, miR-346-5p, miR-495-3p, miR149-5p, miR-485-5p, miR-383-5p, having a decrease in expression more than 2-fold in 2 and 18 months rats were selected (MIMAT0000219, MIMAT0000381, MIMAT0000237, MIMAT0000597, MIMAT0003456, MIMAT0000159, MIMAT0003128, MIMAT0000748).

To test the competence of miRNA for inhibition of EphB2, Luciferase assay an analysis method was used. First, the luciferase-EphB2-3′ UTR reporter construct (Promega) represented by the base sequence of SEQ ID NO: 4 and the candidate miRNA mimic were transfected into HEK293 cells.

The cells were inoculated into 24 wells so as to be 20,000 cells/well and cultured for 24 hours. luciferase-EphB2-3′ UTR reporter and miRNA mimic were mixed with 2 μl of lipofectamine so as to be 0.5 μg, respectively, and were transfected for 4 hours. Then 50 μg of any luciferase reaction solution was added to each well, and luciferase activity was measured using Promega instrument.

As shown in FIG. 3, it was confirmed that only miR-204-5p represented by the base sequence of SEQ ID NO: 1 inhibits EphB2 activity.

To more particularly understand the miR-204-5p and EphB2 relationships. As a result, mRNA expression of miR-204-5p and EphB2 in the sRNA profile results of Example 1 were examined, as a result, as shown in FIG. 4, it was confirmed that mRNA expression of miR-204-5p and EphB2 are inversely correlated as aging progresses.

[Example 5] Determination of miR-204-5p Specificity for EphB2

In the present disclosure, to confirm whether miR-204-5p was specific to EphB2, the activity of wild type EphB2-3′UTR-WT (SEQ ID NO: 2) and The EphB2-3′UTR-MT (SEQ ID NO: 3) in which the miR-204 binding site was point mutated was compared.

The luciferase assay was performed in the same manner as in Example 4, and a scramble mimic (5′GGUUCGUACGUACACUGUUCA3′) was used as a control group.

As shown in FIG. 5A, when EphB2-3′UTR-WT and miR-204-5p were reacted, it was confirmed that the activity of EphB2 was abruptly decreased, on the other hand, there is no activity change in the case of EphB2-3′UTR-MT.

To confirm the change of EphB2 activity according to the miR-204-5p concentration, Transformation was performed in the same manner as above for 0 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM concentrations, and the luciferase activity was measured, and the control group scramble was treated with 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 0 nM to make the amount of material to be transformed equal.

As a result, it was confirmed that the activity of EphB2 was decreased depending as a miR-204-5p concentration-dependent manner.

[Example 6] Confirmation of Change in EphB2 Protein Expression by miR-204-5p

In the present disclosure, change in EphB2 protein expression y miR-204-5p was confirmed using western blot.

First, the dissociated hippocampal nerves were isolated from wild-type rat pups (stage P0). Neurons were inoculated on poly-lysine coated cover glasses (glass coverslips, 18 mm; Bellco Glass, USA) in 12-well plates, and then were maintained in Neurobasal medium containing B27 (Invitrogen, USA), glutamine (Sigma-Aldrich, USA) and penicillin-streptomycin (Sigma-Aldrich, USA), and then 70,000 cells were inoculated into each well, and then cultured in condition of 5% CO2, and 37% humidity.

The miR-204 mimic and miR-204 inhibitors (AGGCAUAGGAUGACAAAGGGAA) were transformed on day 14 of culture, and the calcium phosphate method was used for transformation of neurons.

Cells were cultured in condition of 5% CO₂ and 37° C. with moisture for 7 days after the transformation, cells were obtained, and the cells were agitated for 4 to 60 minutes using a RIPA buffer containing protease inhibitor to dissolve the cells. And the cells were briefly sonicated twice and centrifuged at 13,000 rpm for 4 to 15 minutes to separate the proteins. The separated proteins were subjected to western blotting in the same manner as in Example 3-2.

As a result, as shown in FIGS. 5C and 5D, it was confirmed that the expression amount of EphB2 was significantly decreased to 50% by miR-204, and when the miR-204 inhibitor was treated, it was confirmed that the expression amount of EphB2 was recovered to 130%. In other words, it was confirmed that EphB2 was a direct target of miR-204-5p in the present disclosure.

[Example 7] Regulation of Dendritic Spine Density Through EphB2 Inhibition of miR-204-5p

In the present disclosure, the relationship between miR-204-5p and EphB2 in synaptic plasticity was examined.

First, 1) scramble mimic, 2) miR-204 mimic, 3) EphB2-3′UTR-WT and miR-204 mimic, 4) EphB2-3′UTR-MT and miR-204 mimic were each transformed with the hippocampal nerves cultured for 14 days in the same manner as in Example 6, and then dendritic spine density was observed using a method for immunocytochemistry.

Neurons were treated with 4% paraformaldehyde/4% sucrose PBS and fixed at room temperature for 10 minutes. Cells were washed three times with PBS, treated with PBS containing 0.1% triton X-100 and permeabilized at room temperature for 10 minutes, blocked with 5% BSA in PBS, treated with PBS containing 5% BSA and blocked. Then, the cells were treated with Rabbit anti-GFP (Abcam: 1:1000) first antibody and reacted overnight at 4, washed three times with PBS containing 1% BSA, and treated with Alexa-488 conjugated second antibody (1:1,000, Life technologies) and reacted at room temperature for 1 hour.

Transformed neurons were randomly selected for quantitative analysis from each coverslip in multiple assays, and images were captured with LSM 760 using a 63× object glass. Dendrites were quantitatively analyzed from 50-200 μM cell body. Only second and third dendrites were used for quantitative analysis. All morphological experiments were repeated at least three times (n=10-20 individual experiments). The protrusion, which contained a bulbous head larger than the base, was measured with protrusions of the vertebral-head.

As shown in FIGS. 6A and 6B, it was confirmed that the over-expression of miR-204-5p decreased the spine density by 40% when compared with the control group scramble mimic (3.4 spine/10 um length, including 5.2 spine protrusion, p<0.001).

When EphB2-3′UTR-WT and miR-204 mimic were simultaneously transfected (3.9 spine/10 um length, p>0.5), it was confirmed that there was no significant increase in spine density compared to miR-204 mimic (3.6 spine/10 um length, p<0.001) only infected neurons, on the other hand, when EphB2-3′UTR-MT and miR-204 mimic were simultaneously transfected (5.4 spine/10 nm length, p<0.001), it was confirmed that there was spine density of normal levels.

[Example 8] Regulation of Cell Surface Expression of NMDA Receptor by EphB2 Inhibition of miR-204-5p

Since EphB2 is known to mediate NMDA receptors expressed on the cell surface of neurons, whether miR-204-5p affects the transport and location change of NMDA receptor subunits in hippocampal nerves were examined.

First, it was prepared that 1) scramble mimic, 2) miR-204 mimic, 3) EphB2-3′UTR-WT and miR-204 mimic, 4) EphB2-3′UTR-MT

miR-204 mimic were each transformed with the hippocampal nerves cultured for 14 days in the same manner as in Example 6, and cultured in condition of 5% CO2 and 37% for 7 days.

Biotinylation assay need to be performed to analyze proteins expressed on the cell surface.

The transformed mouse first hippocampal nerves were washed twice with cold PBS solution on ice, and cells were reacted on ice for 30 minutes in PBS solution containing 1 mg/ml sulfo-NHS-SS-Biotin (Pierce Protein Research Products). And then, to remove unattached biotin, the cells were washed with PBS solution containing 100 mM glycine, washed again with PBS solution, and the cells were agitated for 4 to 60 minutes using a RIPA buffer containing protease inhibitor to dissolve the cells. And the cells were briefly sonicated twice and centrifuged at 13,000 rpm for 4 to 15 minutes

The total protein concentration was determined by taking 10% of the cell lysate and the remaining cell lysate (about 200 μg) was reacted with avidin agarose beads (Pierce, USA) for 25 to 60 minutes.

The separated proteins were washed 3 times with PBS, boiled with 2×sample buffer, and then subjected to western blotting with 20 μg of surface protein and total soluble protein.

The blots were hybridized with first antibody and then treated with second antibody, and each antibody was diluted in a Tris-buffered saline solution containing 5% dry milk. Hybridized bands were confirmed using an enhanced chemiluminescence (ECL) detection system (Describe the used ECL solution and measuring instrument information).

α-Tubulin (AbClon; AbC-2001) and NMDA receptor subunit NR1 (Life technologies; 32-0500) were used as the first antibodies and a peroxidase-conjugated second antibody (Jackson ImmunoResearch Laboratories) was used.

Data quantitative analysis was normalized to control group (control culture) using NIH ImageJ software and the expression ratios of total protein and biotin labeled proteins were compared.

As a result, as shown in FIGS. 7A and 7B, it was confirmed that the expression ratio of NR1 protein was significantly decreased (P<0.05, n=3) on neuronal cell surface by miR-204, on the other hand, it was confirmed that the expression ratio of NR1 protein was significantly increased (P<0.05, n=3) by the miR-204 inhibitor.

In addition, to confirm whether the aging affects the protein expression of the NMDA receptor NR1 subunit, western blotting was performed in the same manner as above using the protein sample of Example 3-2.

As a result, as shown in FIGS. 7C and 7D, as a result of confirming the expression of the NMDA receptor NR1 subunit from the young (of 2 months mouse) and the old (of months mouse) hippocampal tissues, It was confirmed that the expression of total NR1 protein was decreased by about 50% in the aging process. The above means that the expression of neurons' cell surface and total NR1 protein was decreased as the expression of EphB2 in neurons is decreased by miR-204,

Example 9: Confirmation of Reduction in Amyloid Plaque Levels by miR-204 Knockdown

In order to identify effect of mir-204 knockdown on the cognitive function, 8-month-old forty SXFAD transgenic male mice (20 mice per group) were used, and each of AAV TD (Tough Decoy) mir-204 and AAV TD control was injected into the hippocampus DG thereof. After 4 months, mice were sacrificed and their brains were dissected for cryodissection (5 mice per group). SXFAD male mice were perfused and fixed with a 4% paraformaldehyde (PFA) solution, and brain tissues thereof were rapidly frozen using a cryostat and sectioned to a thickness of 10 μm. At room temperature, PBS containing 0.2% Triton X-100 and 0.5% BSA was allowed to pass through sections for 1 hour, and then incubation was carried out with primary antibody (that is, 4G8) overnight at 4° C. The sections were then washed 3 times with 0.5% BSA and incubated with secondary antibodies (Alexa Fluor 555 or Alexa Fluor 488, 1:500, Molecular Probes, Eugene, Oreg., USA) at room temperature for 1 hour. Brain sections were mounted on a gelatin-coated cover glass and covered with a DAPI-containing mounting solution (Vector Laboratories, Burlingame, Calif., USA). Images of stained tissue sections were captured using confocal microscopy (TI-RCP, Nikon, Japan) and immunoreactivity thereo was analyzed using ImageJ software. To measure the knockdown effect of miR-204 on hippocampal plaque levels, a total of 10 male 5×FAD mice were used in which each group consisted of 5 mice. Amyloid plaques in the hippocampus were quantified using 6 slices of each brain from −1.70 mm to −2.30 mm with respect to bregma in stereotactic coordinates. The number of Aβ plaques in the hippocampus of AAV TD mir-204 or AAV TD control was determined. Plaques were visualized using antibody 4G8 that recognizes Aβ plaques.

As a result, as shown in FIGS. 8A and 8B, in the case of the AAV TD mir-204 injection group, the level of 4G8-immune response plaques was significantly reduced by 45% and 55%, respectively, in both the CA1 and DG regions of the hippocampus, compared to the AAV TD control group. (FIG. 8B, p<0.001, AAV TD control, n=5 mice; AAV TD miR-204, n=5). These results indicate that knockdown of mir-204 may modulate amyloid plaque load in the hippocampus.

Example 10: Confirmation of Reduction in Human Aβ Level by miR-204 Knockdown

It was tested whether the knockdown of miR-204 in the hippocampus of SXFAD mice could alter soluble human Aβ levels when AAV TD mir-204 or AAV TD controls were injected into 20 mice per group. Human Aβ ELISA was performed using the soluble fraction of hippocampal lysate obtained from mice.

Specifically, three right hippocampus in each group were weighed and homogenized on ice with a 5-fold volume of a mixed solution including 5M guanidine hydrochloride, 50 mM tris hydrochloric acid (pH 8.0) and 1 mM PMSF. Then, hippocampus was centrifuged at 16,000 r/min and 4° C. for 20 minutes to obtain a supernatant. The obtained samples were analyzed for Aβ340 and Aβ342 using the human Aβ340 (catalog number KHB3481) and Aβ342 (catalog number KHB3441) kits from Invitrogen (Carlsbad, Calif.) according to the manufacturer's protocol. The samples were diluted with standard dilution buffer, and 1 ul of protease inhibitor cocktail (Sigma catalog number P8340) was added thereto, and then transferred to an AP antibody coated ELISA plate. Human Aβ340 or Aβ342 detection antibody was added to each well and incubated for 3 hours at room temperature. After incubation, the wells were washed and incubated with anti-rabbit IgG HRP followed by a chromogen solution for 30 minutes each. The reaction was stopped and the absorbance at 450 nm was determined to calculate the Aβ concentration of the sample from the standard curve.

As a result, as shown in FIG. 9, the AAV TD mir-204 group showed a significant decrease in both human Aβ 42 and 40 levels compared to the control (FIG. 9, p<0.01, AAV TD control, n=3 mice; AAV TD miR-204, n=3). These results indicate that miR-204 knockdown may promote the reduction of human Aβ levels.

Having described specific portions of the present disclosure in detail, it will be apparent to a person having ordinary skills in the art that this specific description is merely a preferred embodiment and that the scope of the disclosure is not limited thereby. It is therefore intended that the scope of the disclosure be defined by the claims appended hereto and their equivalents. 

What is claimed is:
 1. A method of treating cognitive impairment or Alzheimer's disease, the method comprising administering, to an individual in need thereof, an effective amount of an agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p.
 2. The method of claim 1, wherein the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p.
 3. The method of claim 2, wherein the nucleic acid molecule is RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2′-O modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate-backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide.
 4. The method of claim 1, wherein the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p comprises an oligonucleotide sequence of SEQ ID NO:
 1. 5. The method of claim 1, wherein the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p comprises a vector containing the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p, or a cell transformed thereby.
 6. The method of claim 5, wherein the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p is included in a viral vector.
 7. The method of claim 6, wherein the viral vector is an AAV vector.
 8. A method of inhibiting amyloid beta comprising: administering, to an individual in need thereof, an effective amount of an agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p.
 9. The method of claim 8, wherein the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p is a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p.
 10. The method of claim 9, wherein the nucleic acid molecule is RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2′-O modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate-backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide.
 11. The method of claim 8, wherein the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p comprises an oligonucleotide sequence of SEQ ID NO:
 1. 12. The method of claim 8, wherein the agent which inhibits the expression of miR-204-5p or an activity of miR-204-5p comprises a vector containing a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p, or a cell transformed thereby.
 13. The method of claim 12, wherein the nucleic acid molecule capable of binding to all or part of the nucleotide sequence of miR-204-5p is included in a viral vector.
 14. The method of claim 13, wherein the viral vector is an AAV vector. 